1. Field of the Invention
The present invention relates to regulation of gene expression in eukaryotic cells. More particularly, the invention relates to a novel and natural antisense RNA negative regulatory system utilizing a genetic element which provides a mechanism by which antisense transcripts are generated.
2. Description of the Background and Related Art
The general mechanism by which transcription of eukaryotic genes is initiated typically involves interaction between several factors including promoters (including initiator elements), enhancers, DNA-binding proteins, and a transcriptional complex comprising an RNA polymerase and associated transcription factors. Typically, an AT-rich region, the TATA motif, is positioned upstream from the start of transcription and is necessary for many promoters to initiate transcription by an RNA polymerase efficiently and accurately. In promoters with or without a TATA box, initiator elements can serve to orient the transcription factors for RNA polymerase II. However, the rate of initiation of transcription is determined by one or more DNA-binding proteins that recognize promoter/enhancer elements in the proximity of the initiation of transcription complexes. Presumably, a DNA-binding protein can influence either the initiation of transcription complexes or the propensity of the complexes to elongate once initiated.
The 5' long terminal repeat (LTR) of the human immuno-deficiency virus (HIV) is a prototypic enhancer-promoter unit containing a standard TATA box, an initiation site (Rittner et al., 1995, J. Mol. Biol. 248:562-580), and upstream elements (e.g., Sp1 and NF-.kappa.B) that are commonly found in many viral and cellular genes, and which are influenced by viral and cellular DNA-binding proteins (see, e.g., review by Jones, 1989, New Biologist 1:127-135). From the HIV double-stranded intermediate, and from the HIV promoter located in the 5' LTR, mRNAs of plus strand polarity are transcribed from minus strand (also called "template") DNA (see Definitions section herein). Depending on the transcript, the MRNA may then be translated into one or more viral proteins including Gag, Pol, Vif, Tat, Vpu, Vpr, Rev, Env, and Nef.
Effective transcription from the HIV promoter is dependent on the presence of Tat for transcriptional activation that dramatically increases the levels of viral mRNAs. In the absence of Tat, predominately short mRNA transcripts are transcribed from the minus strand DNA. These short transcripts terminate near a cis-acting element, the transactivation-responsive region (TAR; Selby et al., 1989, Genes Dev. 3:547-558). Also in the absence of Tat, transcribed is a low basal level of viral mRNAs. Tat function is mediated through TAR, located downstream from the initiation site for transcription. The TAR region, present on a transcript, folds in an energetically favored RNA secondary structure or RNA-stem loop structure (see FIG. 1) which acts as a binding site for Tat. It has been demonstrated that in the absence of Tat, the majority of polymerases that have initiated transcription prematurely disengage from the template. Upon binding of TAR by Tat, Tat acts independently of other promoter elements to stimulate elongation (Rittner et al., 1995, supra). Initiator elements (INRs) have been reported to be necessary for efficient initiation of transcription in mammalian cells in the absence of a TATA box (Javahery et al., 1994, Mol. Cell Biol. 14:116-127; Smale and Baltimore, 1989, Cell 57:103-113). There is uncertainty as to why basal (Tat-independent) transcription from the HIV LTR in vivo is relatively low, and whether there is some repression mechanism affecting the HIV 5' LTR.
A possibility was raised that the plus strand of the viral DNA contains a long open reading frame (ORF), located in the region of the genome complementary to the env gene sequence, that may encode a viral protein (Miller, 1988, Science 239:1420-1422). However, it is not apparent whether this possibility was confirmed, such as by the demonstration of the putative protein or its respective mRNA. The possibility that bidirectional transcription occurs in HIV was further evaluated by Michael et al. (1994, J. Virol. 979-87). It was discovered that a weak negative strand promoter in the U3 region of the 3'LTR of HIV is responsible for the production of RNA transcripts of negative strand polarity. What role (if any) such RNA transcripts, produced from the weak negative strand promoter in the 3' LTR, have in the HIV life cycle remains to be elucidated.
Pathways by which viral or cellular gene expression are regulated (whether involving transcription and/or translation) are targets for intervention. More particularly, the regulation of production of recombinant proteins for industrial or medical applications is a common goal of the biotechnology industry. Using HIV as an example, the transactivation functions have been used to develop HIV-specific and sensitive bioassays (Felber and Pavlakis, 1988, Science 239:184-7), and to evaluate the effects of drugs on the ability of HIV to infect and replicate (see, e.g., Schwartz et al., 1989, Proc. Natl. Acad. Sci. USA 86:7200-7203). Several approaches have been attempted for the antisense inhibition of cellular or viral gene expression. For example, antisense inhibition of HIV replication has met with variable degrees of success in inhibiting some function of the virus. Sites in HIV that are targeted for antisense inhibition include the LTR, the U5 region, the U3 region, the R region, the primer binding site region, the AUG start codon region, the polyP region, RNA splice sites, the leader region, the tat splice site, the rev splice site, and the cap site (see, e.g. U.S. Pat. No. 5,580,761 to Greatbatch et al.). It has also been suggested that synthesized oligonucleotides antisense to the TAR stem-loop may be capable of disrupting the secondary structure of the TAR stem-loop (Vickers et al., 1991, Nucleic Acids Res. 19:3359-3368; U.S. Pat. No. 5,512,438 to Eckers), or antisense to tat MRNA (U.S. Pat. No. 5,166,195 to Ecker), thereby ultimately disrupting transactivation mediated by the binding of Tat to TAR.
Accordingly, there has been and continues to be a long-felt need for the intracellular production of antisense molecules which are capable of effectively functioning to regulate gene expression at either a transcriptional and/or translational level. Desirably, such an approach to antisense therapy would employ, and thereby "turn up" a mechanism of control of transcription and/or translation that may be found intrinsically in mammalian cells.